Many enzymes containing redox-active metal centers play significant roles in cellular function, and are often involved in a variety of physiologically important processes. In particular, several Mn-containing metalloproteins have emerged with functional roles in O{2} metabolism since the identification of Mn as an essential metal in biological redox catalysis. These include a mitochondrial Mn-superoxide dismutase (SOD2) that detoxifies superoxide radicals into O{2} and peroxide; a non-heme Mn-containing pseudocatalase that catalyzes the decomposition of peroxide into H{2}O and O{2}; and the oxygen-evolving complex (OEC) in photosystem II (PSII), which is possibly the most important due to its key role in the oxidation of H{2}O to O{2} during photosynthesis. Nearly all of the atmospheric O{2} that supports aerobic life is produced and replenished by the OEC through H{2}O oxidation; hence, this light-induced reaction is one of the most important biological redox processes found in nature. Although it is known that the OEC is composed of a heteronuclear Mn4CaOx cluster where four electrons are extracted in a stepwise manner from two H{2}O molecules to produce one O{2} molecule, the detailed structure and mechanism of how this process occurs are not well understood. Furthermore, conventional X-ray crystallography and spectroscopy approaches are limited by the sensitivity of the redox-active metal complex to radiation damage by photoreduction. However, the recent development of the powerfully intense X-ray free electron laser (X-FEL) and application of the collect before destroy approach provide a viable option for overcoming this obstacle. Thus, a key objective of this proposal is to determine the structure of the intact OEC and elucidate the catalytic mechanism by which H{2}O is oxidized to O{2} by mapping the time evolution of the Mn{4}CaO{x} cluster using this new X-FEL technology. Specifically, X-ray diffraction (XRD) and X-ray emission spectra (XES) will be simultaneously measured from a continuous stream of PSII microcrystals with femtosecond X-FEL pulses in order to determine not only the electronic and geometric structure of the Mn{4}CaO{x} cluster, but also the integrity of the metal complex. Two fundamental points that are central to understanding photosynthetic water oxidation include: (i) the temporal evolution of the OEC electronic structure, and (ii) the structural dynamics in the ligand environment and Mn{4}CaO{x} cluster as it cycles through the catalytic steps. To address these points and map the light-induced chemical steps in real time, a combined laser excitation 'pump' and X-FEL 'probe' with variable time delays will be incorporated into the experimental setup. Not only will this study lead to an understanding of the mechanism of H{2}O oxidation to form O{2}, but the methodology developed here should also have broad applications as a model study for using X-FELs to determine structure and dynamics in other physiologically important membrane proteins and redox- active metalloenzymes that are prone to X-ray radiation damage.